Fig. 1. The elution profile of LDH from Blue-Sepharose by NaCl or arginine. The same amount of LDH (1 mg) was applied for each experiment. The flow rate was 0.3 ml/min. Elution solvent for each panel is indicated by an arrow. Different elution positions of the first peak are due to the differences in the time of application of the eluent.

Fig. 2. The recovery of LDH from Blue-Sepharose by NaCl or arginine. One milligram of LDH was applied. Black bar, elution with arginine; white bar, elution with NaCl. The elution experiment using 0.75 M arginine was not performed. Elution recovery was estimated by pooling the eluted fractions and measuring the absorbance of the pool. Each volume of the load and eluted pool was adjusted to equal volume before taking absorbance. The ratio of absorbance of the pool to load was used at percentage recovery.

Fig. 3. The recovery of LDH from Blue-Sepharose by NaCl followed by 1 M arginine elution. One milligram of LDH was applied. Black bar, first elution with NaCl at indicated concentration; white bar, second elution with 1 M arginine; blue bar, total recovery. Elution recovery was estimated by pooling the eluted fractions and measuring the absorbance of the pool. Each volume of the load and eluted pool was adjusted to equal volume before taking absorbance. The ratio of absorbance of the pool to load was used at percentage recovery.

Fig. 4. Linear gradient elution of LDH from Blue-Sepharose by NaCl (left-side panel) or arginine (right-side panel). Four milligrams of LDH were applied. The flow rate was 0.3 ml/min.

Fig. 5. SDS–PAGE analysis of fractions from ATP–agarose column. Lane 1 (molecular weight marker), lane 2 (flow through), lane 3 (buffer wash), lane 4 (0.5 M arginine), lane 5 (second 0.5 M arginine), lanes 6 and 7 (1 M arginine) and lanes 8 and 9 (2 M arginine). The band at 21 kDa corresponds to NDK, as examined by Western blotting analysis using an anti-NDK(Ha293) antibody (MI and MT, unpublished results).